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1 Journal of Bacteriology and Virology Vol. 42, No. 4 p Review Article Cellular and Systemic Interactions of Orientia tsutsugamushi with Mammalian Host Se-Yoon Kim, Myung-Sik Choi and Nam-Hyuk Cho * Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi infection and one of main causes of febrile illness in the Asia-Pacific region. It has been estimated that one billion people are at risk and one million new cases arise each year in the endemic region. Despite of aggressive attempts to develop a prophylactic vaccine against scrub typhus during last several decades, all approaches have failed to generate long lasting immunity. In addition, little is known about the immunological pathogenesis of scrub typhus. In this review, we summarized recent findings of cellular and systemic interaction of O. tsutsugamushi with mammalian host, especially focusing on the molecular basis of intracellular invasion and immunological changes observed during the bacterial infection. Key Words: Orientia tsutsugamushi, Scrub typhus, Immune responses, Intracellular invasion 서론쯔쯔가무시병 (scrub typhus) 은세포내절대기생세균인 Orientia tsutsugamushi 균의감염에의해발생하는급성열성질환이다 (1). 야생에살고있는털진드기 (Leptotrombidium species) 에서난소전파 (transovarian transmission) 를통해자연계에서전파되고있으며, 감염된털진드기가사람을물어체액을흡입하는과정에서침샘에존재하던균이인체감염을일으키게된다. 쯔쯔가무시병은우리나라를포함하여일본, 중국, 인도, 동남아시아지역, 그리고호주북부지역에서만발생하고하고있는데, 이는 O. tsutsugamushi를전파하는털진드기종들이이지역에서만서식하기때문인것으로알려져있다 (2). 이지역에서매년약백만명의환자가발생하고있는것으로추정되고있으며 (3), 가을철에만주로환자가발생하는 우리나라의경우, 2000년대초반에환자가급격히증가하여현재는매년약 6천명의환자발생이전국적으로보고되고있다 (4). 최근에는인도 (5), 중국 (6) 등지에서도쯔쯔가무시병의심각한 outbreak가종종보고되고있으며, 기존에는발생이보고되지않았던중국북부지역및중동지역에서도환자발생이확인되고있다 (7, 8). 또한유행지역으로국제여행을다녀온여행객들의쯔쯔가무시병감염도보고되고있어이에대한대비가필요한상황이다 (9). 쯔쯔가무시병은항생제처방을통해쉽게치료가가능하지만, 초기증상이유사한다른급성열성질환들과조기감별진단이용이하지않아항생제치료가늦어질경우심각한전신성염증질환을유발할수있다 (10). 항생제치료를받지못할경우, 유행지역및감염균의유전형, 그리고환자의면역능에따라약 50% 에달하는치사율이보고되기도하였으며 (11), 항생제치료효과가잘나타나 Received: October 2, 2012/ Revised: October 26, 2012/ Accepted: October 30, 2012 * Corresponding author: Nam-Hyuk Cho. Department of Microbiology and Immunology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul , Korea. Phone: , Fax: , chonh@snu.ac.kr ** This study was supported by grants (A111503) from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( 276

2 Cellular and Systemic Interactions of Orientia tsutsugamushi with Mammalian Host 277 지않는임상예들도보고되고있어백신개발의필요성이지속적으로제기되고있다 (12). 쯔쯔가무시병환자의임상적특징으로는발열, 오한, 두통과전신쇠약감이나타나며, 우리나라의경우약 90% 의환자에서털진드기에물린자리에가피가관찰된다 (13). 발병초기에적절한항생제치료를받지못할경우, 폐렴, 급성신부전, 뇌수막염, 뇌염, 위장관출혈등, 다양한장기의기능부전이나파종성혈관내응고를유발하여사망에이르게한다 (14). 이렇게다양한장기에서병리학적변화를유발하는이유는다른리케치아균들과마찬가지로 O. tsutsugamushi 균이혈관내피세포에감염하여국소적인또는전신성혈관염을일으키는것으로알려져있기때문이다 (15). 하지만최근환자의가피를면역염색법으로조직검사한결과, 이세균이주로활성화된큰포식세포와수지상세포에주로감염되어있고혈관내피세포에는거의감염되어있지않은사실이보고되었다 (16). 생체외실험실감염이나실험동물감염에서는큰포식세포 (17), 중성구 (18), 수지상세포와같은포식세포뿐만아니라포식작용이없는혈관내피세포 (19) 나섬유모세포, 상피세포 (20) 에도감염되어활발하게증식할수있다. 쯔쯔가무시병의병인기전은생체외실험이나생쥐감염모델을이용해주로연구되어오고있으며, 백신개발연구가오랜기간시도되어왔다. 하지만, 아직까지효과적인백신이개발되지못하고있는데, 이는이세균의주요막항원인 TSA56 단백질의유전적변이가매우다양하고, 사람을포함한영장류에서백신에의한면역기억이 1년이상지속되지못하여방어면역을유도할수없었기때문이다 (12). 본리뷰에서는최근발표된연구논문들을통해밝혀진 O. tsutsugamushi에의한병인기전을정리하였다. O. tsutsugamushi의숙주세포침입기전세포내절대기생세균인 O. tsutsugamushi는진핵세포의세포질내에서이분법을통하여복제된다. 이를위해숙주세포의세포질내로침입하는과정을거쳐야하며, 전자현미경관찰을통한이전에보고에의하면, 부착 (adhesion), 포식유도 (induced phagocytosis), 그리고탈엔도솜 (endosomal escape) 과정을거쳐세포질내로이동한다 (21). 포식된세균들은감염 2시간내에 ph 변화에의존적 으로엔도솜에서이탈하여세포질로유리되며 (22), 탈출한세균들은미세관 (microtubule) 구조에결합하여핵주변에존재하는 microtubule-organizing center 로이동하여복제한다 (23, 24) (Fig. 1). 현재까지밝혀진 O. tsutsugamushi 의세포수용체는 heparan sulfate proteoglycans (HSPGs) 과 integrin 분자가있다. HSPGs는당화된세포막단백질로, heparan sulfate glycosaminoglycan이단백질과결합된형태로다양한세포표면에발현되어있다. Heparan sulfate의합성이결손된돌연변이 Chinese hamster ovarian 세포주는 wild type 세포주에비해 O. tsutsugamushi에훨씬덜감염되는것이확인되었다 (25). 또한감염배지에 heparan sulfate나 heparin을첨가해주면농도의존적으로이세균의숙주세포감염을억제하는것이관찰되었으며, 35 S -labelled heparin이 O. tsutsugamushi 입자에결합하는것도확인되었다 (25). Syndecan 단백질은 HSPGs 중에서포유동물세포의표면에가장많이발현되어있는것으로, 이단백질중에서특히 Syndecan-4가 O. tsutsugamushi의세포내침입에관여하는것이확인되기도하였다. Syndecan-4 를과발현하거나발현을저해한 REF 세포주에서이세균의숙수세포침입이 Syndecan-4의발현양에비례하여증가하는것이보고되었으며, 재조합 Syndecan-4 단백질을감염배지에첨가하였을때 O. tsutsugamushi의감염을억제하는것도관찰되었다 (26). 최근에는 HSPG인 Syndecan-4 이외에도 integrin α5β1 분자가 O. tsutsugamushi 의감염과정에관여하는것이확인되었다 (20). O. tsutsugamushi가 HeLa 세포주에감염될때 integrin α5β1 분자와 co-localization하는것이관찰되었으며, 감염직후 integrin α5β1 의 downstream 신호전달분자인 focal adhesion kinase, src kinase, 및 RhoA GTPase의활성화가유도된다 (20). Protein tyrosine kinase나 RhoA GTPase의활성을억제한경우에는이세균의감염이유의하게감소하였다 (20). 이신호전달체계에의해활성화된신호경로는 O. tsutsugamushi 균의감염부위에서 actin 세포골격재배열을통한 membrane protrusion을유도함으로서비포식세포감염시관찰되는포식유도를유발하는것이증명되었다 (20). 숙주세포의수용체와결합하는 O. tsutsugamushi의세균리간드에대한연구도최근진전을보이고있다. 이세균의주요막단백질인 TSA56 단백이세포외기질단백질인 fibronectin과결합하는것이보고되었는데, 이러한결합이세균의세포내침입을유의하게증가시키는것이확

3 278 S-Y Kim, et al. OT Fibronectin Syndecan-4 Talin Integrin α5β1 Paxillin RhoA Clathrin Actin FAK Src Figure 1. Hypothetical model for the intracellular invasion of O. tsutsugamushi. O. tsutsugamushi (OT) surface molecules (e.g., TSA56 and ScaC) mediate bacterial adhesion to host cells by binding to host-specific receptors, such as Syndecan-4 and fibronectin. The interaction between O. tsutsugamushi and fibronectin may mediate the engagement of integrins, which subsequently may activate downstream signaling molecules, such as FAK, Src kinase, and RhoA GTPase. Signaling adapters, such as talin and paxillin, are recruited to the site of infection. These signaling events consequently induce the internalization of O. tsutsugamushi into non-phagocytic host cells, which involves the clathrin-mediated endocytic pathway. The bacteria subsequently travel through the early and late endosomes and escape from the endosomal compartment within 2 h after infection. Cytosolic O. tsutsugamushi might move to microtubule organizing center via trafficking thorough microtubules and replicates in the perinuclear region (24). 인되었다 (27). Fibronectin과 TSA56의결합은이세균막단백질의 C-terminal region (aa ) 에의해매개되며, 이부위를포함하는재조합단백질은농도의존적으로 O. tsutsugamushi의세포내침입을억제하였다. 특히 TSA56 의아미노산 부위가 fibronectin과가장강한결합력을보였으며, 세포내침입억제효과도가장높게나타났다 (20). TSA56 이외에도이세균이보유하고있는 surface cell antigen (Sca) 단백질들중에서 ScaC 단백질이숙주세포부착에관여하는것이보고되었다 (28). 흥미롭게도 ScaC 단백질이 TSA56과마찬가지로 fibronectin과결합하는것이확인되었으며 (28), 이러한결과들을토대로하였을때, O. tsutsugamushi는숙주의 fibronectin 및 integrin 분자들을포유류의숙주세포부착과침입에활용하는것으로보인다. 이러한세균의리간드들과숙주세포수용체들의상호작용은다양한신호전달체계의활성화를통하여이세균의숙주세포침입에관여할것으로추정되고있으므로이에대한후속연구를통하여보다심 도있는침입기전연구가진행되어야할것이다. O. tsutsugamushi 감염에대한선천면역반응 O. tsutsugamushi는피부감염후림프계를통해전신으로퍼져나갈것으로추정되고있었다. 그러나급성감염환자의혈액에존재하는단핵구에서이세균이감지되기때문에직접적으로혈행을통해전신으로퍼져나갈가능성도높다 (29). O. tsutsugamushi 감염에대한선천면역반응연구는비교적기초단계에머무르고있는실정이지만, 최근들어몇가지흥미로운연구결과들이발표되고있다. 우선 O. tsutsugamushi 균에대한방어면역반응에서가장중요할것으로여겨지는세포인큰포식세포의감염에대한반응이이전부터보고되었다. O. tsutsugamushi 에감염된큰포식세포인 J774A.1 세포주에서전사인자인 NF-κB의활성화와 MIP-1α/β, MIP-2, 그리고 MCP-1 등의케모카인발현증가가보고되었다 (17). 이케모카

4 Cellular and Systemic Interactions of Orientia tsutsugamushi with Mammalian Host 279 인들은감염부위로다른염증세포들을, 특히단핵구와림프구를불러모아세균을제거하는데중요한역할을할것으로생각된다. 큰포식세포의활성화를유발하는세균인자는아직밝혀지지않았으며, 이전보고에따르며, 열에안정한세균성분이세포를자극하는것으로추정하고있다 (17). Extracellular signal-related kinase (ERK) 와 p38 mitogen-activated protein (MAP) kinase들을특이적으로억제하는화합물들을처리하면케모카인의발현에영향을미치지않는것으로보아 MAP kinase 신호경로는큰포식세포에서케모카인발현에크게영향을미치지않는것으로보인다. 그러나, cytochalasin D 처리가케모카인발현을억제하므로이세균의세포내이입이큰포식세포에서의케모카인발현에필요한것을확인한바있다 (30). 케모카인들외에도 O. tsutsugamushi가감염된큰포식세포에서는 TNF-α, IFN-β, IL-1β와같이선천면역반응에중요한싸이토카인들이발현된다 (30, 31). 이전보고에따르면, TNF-α나 IFN-γ가처리된큰포식세포는 O. tsutsugamushi 감염에의한세포사멸을막을수는없지만세균의세포내복제를유의하게감소시킬수있다 (31). 큰포식세포에서발현되는케모카인들과달리, MAP kinase 를억제하면이싸이토카인들의발현은줄어드는것이확인되므로 TNF-α와 IFN-β의발현은 MAP kinases 신호경로에의존적이다 (29, 30). 또한 IFN-β의발현이 cytochalasin D에의해완전히억제되는데, 이는케모카인과마찬가지로세균의숙주세포내이입과정이 IFN-β의발현에필수적임을보여준다 (30). IL-1β의경우, 살아있는세균만이큰포식세포에서그발현을유도할수있으며, IL-1 수용체신호전달경로는 O. tsutsugamushi 감염에대한효과적인방어면역에매우중요한것으로보고되었다 (33). O. tsutsugamushi 감염에의한 IL-1β 발현은 ASC inflammasome 활성화와 MyD88 신호경로에의존적이라는사실이 knock-out mouse 유래의큰포식세포감염실험을통해확인되었으며, 이는 IL-1β가발현되기위해서는 Toll-like receptor 신호전달경로의활성화와 inflammasome 의활성화모두를필요로한다는것을의미한다 (33). 큰포식세포와더불어혈관내피세포도 O. tsutsugamushi 감염에의해다양한케모카인과싸이토카인을발현하는것으로보고되었다. MCP-1, IL-8, 그리고 RANTES (regulated upon activation, normal T-cell expressed, and secreted) 등의케모카인발현이 HUVEC (human umbilical vascular endothelial cell) 세포주와 HMEC (human dermal microvascular endothelial cells) 세포주에서감염에의해유도되며, 이러한발현증가는 AP-1 전사인자의활성화에의존적이다 (19, 34). ECV304 세포주감염실험에서는 IL-1α/β, IL-6, IL-8, IL-10, IL-15, IL-32, TNF-α/β의발현이감염에의해증가하는것이확인되었다 (35). 이보고에따르면, O. tsutsugamushi 감염에의한싸이토카인들의발현증가는 NOD1 (nucleotide-binding oligomerization domaincontaining protein 1) knockdown에의해감소하는데, 이러한감소는다시 IL-32의처리에의해회복될수있다. 따라서 ECV304 세포주에서는 NOD1 신호경로에의한 IL- 32의발현이다른염증성싸이토카인들의발현을조절하고있음을시사하고있다 (35). NOD-like receptor (NLR) family에속하는 NOD1은펩티도글리칸에서유래한 γ-dglutamyl-meso-diaminopimelic acid (meso-dap) 를인지하여활성화되는것으로알려져있는데, O. tsutsugamushi 균의유전체분석에의하면이성분을합성하는유전자들이거의존재하는것으로확인되었기때문에숙주세포의세포질로이동한세균은 NOD1에의해인지되어싸이토카인분비를유도하는것으로추정할수있다 (36, 37). 큰포식세포와혈관내피세포외에도선천면역과적응면역을연결하는데중요한역할을담당하는것으로알려진수지상세포가 O. tsutsugamushi에감염되는사실이쯔쯔가무시병환자의가피조직검사를통해최근보고되었다 (16). 피부감염에의한수지상세포의활성화및림프절로의이동은이세균에대한적응면역반응결정하는데매우중요하기때문에, 이에대한후속연구가필요한상황이며, O. tsutsugamushi 감염에의한수지상세포의활성조절기전연구는쯔쯔가무시병의면역병리를이해하는데중요한단초를제공할것이다. O. tsutsugamushi 감염에대한적응면역반응과면역체계의변화 O. tsutsugamushi 감염에대한방어면역기전은생쥐감염모델에서주로연구되어왔는데, 여러연구보고를통해세포매개면역반응이매우중요하다고알려졌다 (12). 항원특이적 T 림프구들이큰포식세포의활성화를유도하여강력한세포매개면역반응을유도함으로써감염된세균들을제거할수있다 (38, 39). 항원특이적 T 림프구의중요성은 IFN-γ를분비하는 T H1 림프구의수동전달을통한방어면역능획득시험을통해서도확인되었다

5 280 S-Y Kim, et al. Table 1. Summary of leukocyte changes observed during acute and convalescent phase of scrub typhus (52). Acute Convalescent Neutrophil Increase - Monocyte - - Lymphocyte Decrease - CD4 + T CD8 + T CD4 + T CD8 + T Decrease - Decrease Increase Apoptotic Increase Increase - Increase Proliferative Increase Increase - Increase Type 1 Decrease Decrease - - Type Treg Decrease Decrease IL-7Ra low Increase Increase PD-1 high Increase Increase (40). 또한쯔쯔가무시병환자에서활성화된 cytotoxic CD8 T 림프구들이증가하는것이관찰되었는데, 이는 O. tsutsugamushi에감염된세포들을제거하는중요한역할을할것으로추정된다 (41). 이러한세포매개면역뿐만아니라항체에의한체액면역반응도 O. tsutsugamushi의숙주세포침입을차단하는등의중화반응을통해방어면역능에기여하는것으로알려져있다 (12). 하지만항체에의한방어면역은이세균의다양한혈청형에매우특 이적인것으로보고되었으며 (42), 생성된항원특이적항체의수명이비교적짧고 (43), 불활성화된세균을면역하였을때방어면역능을유발할수있지만항체반응은거의나타나지않는다는사실을고려하였을때 (44), 항체반응자체만으로는충분한방어면역을유도하기어려운것으로생각되고있다. 따라서 O. tsutsugamushi 감염에대한효과적인방어면역능을위해서는항체반응과세포매개면역반응을동시에유도할수있어야한다. O. tsutsugamushi 감염시사람을포함한영장류에서나타나는면역체계의변화는쯔쯔가무시병의면역병리이해뿐만아니라, 백신개발을위한기초전략을수립하는데매우중요한기초지식을제공할수있다. 현재까지수행된연구들에서는주로감염시나타나는혈액내싸이토카인의발현변화그리고백혈구들의아형및분포변화에집중하여왔다. 쯔쯔가무시병을앓고있는대부분의환자들에서공통적으로관찰되는싸이토카인발현변 화는 IFN-γ와 IL-10의증가이다 (45, 46). 이외에도 IL-1β, IL-12, TNF-α, G-CSF, M-CSF 등의싸이토카인이쯔쯔가무시병환자의혈청내에증가되는것이보고되었다 (45~48). 이싸이토카인들의발현변화는환자의감염기간, 병의중증도, 면역능, 그리고감염된세균의혈청형및유전형에따라다르게나타나는것으로추정되고있다. TNF-α의경우병의중증도와상관성이높다는보고가있으나 (45), 그렇지않은결과도이전에발표되었기때문에 (46) 이와관련된보다체계적인연구가필요하다. 한가지흥미로운사실은, 쯔쯔가무시병환자의혈액내 IL-10의발현양과검출된세균 DNA의양이양성적인상관관계를보이며 (46), 혈중세균 DNA의양과쯔쯔가무시병의중증도가매우밀접하게연관되어있다는사실이증명되었기때문에 (49), O. tsutsugamushi 감염에의한 IL-10의발현조절에대한자세한기전연구가이어져야할것이다. 쯔쯔가무시병환자에서나타나는면역세포의변화는이감염질환의병인기전을이해하는데역시중요하다고생각되고있으나, 현재까지소수의관련연구보고가있었다 (41, 50, 51). 최근우리실험실에서는쯔쯔가무시병환자들을급성감염기와회복기로구분하여이때나타나는백혈구들의변화양상을비교분석하였다 (52). 이에따르면, 급성감염환자에서중성구의수가증가하였으며, T 림프구의수가감소하였는데, 특히 CD4 + T 림프구의수가유의하게감소한다 (Table 1). 그리고회복기에는 CD8 + T 림프구의수가증가하였는데, 대부분활성화된표현형을가진 T 림프구들이증가하였다. 흥미롭게도급성감염기에는 T 림프구들의세포자멸사 (apoptosis) 와분열이동시에활발하게일어나는것이관찰되었다. 또한 CD4 + Foxp3 + CD25 ++ regulatory T 림프구들이감염기간동안현저하게줄어드는현상도확인되었다. 이러한면역학적변화는유행지역의쯔쯔가무시병환자들에서재감염이자주관찰되며 (50), 백신접종에도불구하고 O. tsutsugamushi를감염시킨동물감염모델에서일시적인면역억제상태가나타나는것과밀접한관련이있을것으로보인다 (53, 54). 감염초기면역세포의변화들이쯔쯔가무시병의면역병리에어떻게관련되어있는지, 그리고이러한현상들이어떠한기전에의해서발생하는지에대한후속연구가더필요한상황이며, 이를통해밝혀진내용들은쯔쯔가무시병의병인기전을이해하고효과적인백신개발을위한기초자료로활용될수있을것이다.

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract